A pan-variant miniprotein inhibitor protects against SARS-CoV-2 variants

The continued evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has compromised neutralizing antibody responses elicited by prior infection or vaccination and abolished the utility of most monoclonal antibody therapeutics. We previously described a computationally-designed, homotrimeric miniprotein inhibitor, designated TRI2-2, that protects mice against pre-Omicron SARS-CoV-2 variants. Here, we show that TRI2-2 exhibits pan neutralization of variants that evolved during the 4.5 years since the emergence of SARS-CoV-2 and protects mice against BQ.1.1, XBB.1.5 and BA.2.86 challenge when administered post-exposure by an intranasal route. The resistance of TRI2-2 to viral escape and its direct delivery to the upper airways rationalize a path toward clinical advancement.

We previously described a computationally-designed, homotrimeric miniprotein inhibitor, designated TRI2-2, that binds with high avidity to SARS-CoV-2 S as a result of simultaneously engaging all three receptor-binding domains (RBDs) within a S trimer 11,12 .We showed that intranasal administration of TRI2-2 after viral exposure protected mice from challenge with the SARS-CoV-2 Beta and Delta variants 12 .The low manufacturing cost of TRI2-2 along with its antiviral efficacy when delivered to the upper respiratory tract are attractive properties for the development of next-generation countermeasures blocking SARS-CoV-2 at the site of initial infection.
To study the protective efficacy of TRI2-2 in vivo against immune evasive SARS-CoV-2 Omicron variants, we intranasally inoculated highly susceptible K18-hACE2 mice 16 with 10 4 FFU of BQ.1.1,XBB.1.5,or BA.2.86.One day later, we intranasally administered a single 10 mg/kg dose of TRI2-2 or an influenza virus control minibinder 17 .For all variants evaluated, postexposure TRI2-2 treatment protected against weight loss throughout the duration of the experiments and reduced viral titers in the lungs and nasal turbinates six days post-challenge as compared to the control (influenza virus) minibinder (Fig. 2    SARS-CoV-2 breakthrough infections elicit more robust neutralizing antibody titers in the human upper respiratory tract at the site of initial infection, than intramuscular vaccination alone [19][20][21] .These findings, along with waning of binding and neutralizing antibodies, likely contribute to the continued transmission of SARS-CoV-2 globally and motivate the development and evaluation of next-generation countermeasures that may be administered intranasally or orally.Preclinical assessment of intranasally administered influenza and sarbecovirus vaccine candidates demonstrated the induction of lung-resident protective mucosal humoral and cellular immunity at the site of viral entry [22][23][24][25][26] and lipopeptide fusion inhibitors prevented SARS-CoV-2 direct-contact transmission in ferrets 27 .Furthermore, post-exposure prophylaxis nasal spray administration of the SA58 monoclonal antibody in humans was shown to markedly reduce the risk of contracting COVID-19 28 .
The computationally-designed TRI2-2 minibinder mediates pan-variant neutralizing activity and in vivo protection of mice in both the upper and lower airways against the highly immune evasive SARS-CoV-2 BQ.1.1,XBB.1.5,and BA.2.86 variants.These data show that TRI2-2 can accommodate residue substitutions within its epitope and provide a molecular framework to explain the remarkable retained neutralization of variants that have emerged since the pandemic began in 2019.Moreover, TRI2-2 is endowed with exceptional biophysical stability, enabling cost-effective, large-scale microbial production, setting it apart from monoclonal antibodies that are expensive to manufacture and more challenging to scale.TRI2-2 will be evaluated in humans in an upcoming clinical trial and could herald a new era of computationally-designed prophylactics and therapeutics.

Production of recombinant SARS-CoV-2 S RBDs
The SARS-CoV-2 RBDs were expressed in Expi293 cells (Thermo) at 37°C and 8% CO2.Cells were transfected with the corresponding plasmids using Expifectamine (Thermo) following the manufacturer's protocol.Four days post-transfection, supernatants were clarified by centrifugation at 3000 g for 30 minutes, supplemented with 25 mM phosphate pH 8.0, and 300 mM NaCl and then passed through a 0.22µm sterile filter.Supernatants were then bound to 1 mL Histrap excel columns previously equilibrated in 25 mM phosphate pH 8.0, 300 mM NaCl.Nickel columns were washed with 25 mM phosphate pH 8.0, 300 mM NaCl, and 10mM imidazole prior to elution with 25 mM phosphate pH 8.0, 300 mM NaCl and 300mM imidazole.
After buffer exchanging into 20mM phosphate pH 8.0 and 100mM NaCl using a centrifugal filter device with a MWCO of 10kDa, the purified RBDs were biotinylated using the BirA biotin-protein ligase reaction kit (Avidity).The biotinylated RBD's were bound, washed, and eluted again on the same affinity column.Purified biotinylated RBD's were then concentrated and eluted on a Superdex200 increase 10/300 size-exclusion column (Cytiva) equilibrated in 20mM phosphate pH 8.0 and 100mM NaCl.Fractions containing monomeric and monodisperse RBDs were flash frozen and stored at -80°C until use.

Production of recombinant TRI2-2 and influenza virus minibinders
The TRI2-2 and influenza virus minibinders were cloned into pET29b between the NdeI and XhoI restriction sites by Genscript.The TRI2-2 minibinder was cloned with a C-terminal polyhistidine tag and the influenza minibinder was cloned with an N-terminal polyhistidine tag 17 .Minibinders were expressed in Lemo21(DE3) cells (NEB) in TB II Media (MP Bio) at 37°C with IPTG induction.After cell harvest, pellets were resuspended in Gibco dPBS and lysed by microfluidization at 18,000 psi.Whole cell lysates were clarified by centrifugation at 18000 g for 30 minutes and supernatants were then bound to a 5 mL Histrap Nickel Sepharose FF column (Cytiva) previously equilibrated in Gibco dPBS supplemented with 30mM Imidazole.Nickel columns were washed with Gibco dPBS (ThermoFisher) supplemented with 30mM imidazole prior to elution with Gibco dPBS supplemented with 500mM Imidazole.Using a centrifugal filter device with a MWCO of 3kDa, the IMAC fractions containing minibinders of interest were concentrated and then further purified by size-exclusion chromatography using an Superdex S75 Increase 10/300 GL column (Cytiva) equilibrated in Gibco dPBS as running buffer.
Fractions containing TRI2-2 or Influenza minibinders were further concentrated (as needed) filtered with a 0.2 μm filter, and then tested for endotoxin (LAL Charles River) prior to being flash frozen and stored at -80°C until use.

Binding analysis using biolayer interferometry (BLI)
BLI binding assays were performed on an Octet Red (Sartorius) instrument operated at 30 with shaking (1000 rpm).Streptavidin biosensors were hydrated in a 10x kinetics buffer (Sartorius) for 10 min prior to the experiment.Biosensors were incubated in a 10x kinetics buffer for 60s followed by the loading of biotinylated RBDs to the tip, all to a final level of 1 nm.Loaded biosensors were equilibrated in a 10x kinetics buffer for 120s.For affinity binding assays to determine K D values, RBD-loaded tips were dipped into a concentration series of TRI2-2 (3-fold serial dilution from 25 nM to 0.9 nM) for 300 s followed by 300 s of dissociation in a 10x kinetics buffer.Global fits were used to calculate K D values using a 1:1 binding fit model.Data were plotted using GraphPad Prism.Assays were replicated with two biological replicates (recombinant RBD proteins generated on different days) and representative graphs and values are shown in Supplementary Figure 1 and Supplementary Table 1, respectively.
Production of VSV pseudoviruses SARS-CoV-2 spike VSV pseudoviruses were produced using HEK293T cells seeded on BioCoat Cell Culture Dish: poly-D-Lysine 100 mm (Corning).The following day, cells were transfected with spike constructs using Lipofectamine 2000 (Thermo Fisher Scientific) in Opti-MEM transfection medium.After 5h of incubation at 37 °C with 5% CO2, cells were supplemented with DMEM containing 10% of FBS.On the next day, cells were washed with three times with DMEM and infected with VSV (G*ΔG-luciferase) for 2h, followed by five time wash with DMEM medium before addition of anti-VSV G antibody (I1-mouse hybridoma supernatant diluted 1:40, ATCC CRL-2700) and medium.After 18-24 h of incubation at 37 °C with 5% CO 2 , pseudoviruses were collected and cell debris removed by centrifugation at 3,000xg for 10 min.Pseudoviruses were further filtered using a 0.45 µm syringe filter and concentrated 10x prior to storage at -80°C.

Neutralization assays
For SARS-CoV-2 S VSV neutralization with TRI2-2 and AHB2, HEK293T cells with stable human ACE2 expression in DMEM supplemented with 10% FBS and 1% PenStrep were seeded at 40,000 cells/well into 96-well plates [3610] (Corning) coated with poly-lysine [P4707] (Sigma) and incubated overnight at 37°C.The following day, a half-area 96-well plate (Greiner) was prepared with 3-fold serial dilutions of TRI2-2 and AHB2 with a starting concentration of 1 μM.An equal volume of DMEM with diluted pseudoviruses was added to each well.All pseudoviruses were diluted between 1:3 -1:27 to reach a target entry of ~10 6 RLU.The mixture was incubated at room temperature for 45-60 minutes.Media was removed from the cells and 40 μL from each well of the half-area 96-well plate containing minibinder and pseudovirus were transferred to the 96-well plate seeded with cells and incubated at 37°C for 1h.After 1h, an additional 40 μL of DMEM supplemented with 20% FBS and 2% PenStrep was added to the cells.After 18-20h, 40 μL of One-Glo-EX substrate (Promega) was added to each well and incubated on a plate shaker in the dark for 5 min before reading the relative luciferase units using a BioTek Neo2 plate reader.Relative luciferase units were plotted, and normalized in Prism (GraphPad): 0% entry being cells lacking pseudovirus and 100% entry being cells containing virus but lacking minibinder.Prism (GraphPad) nonlinear regression with "[Inhibitor] versus normalized response with a variable slope" was used to determine IC 50 values from curve fits with 3 technical repeats.3 biological replicates were carried out for each samplepseudovirus pair.

Fusion assays
Cell to cell fusion assay using a split-GFP system was conducted as previously described.BHK-21-GFP 1-10 cells were split into 6-well plates at a density of 250,000 cells per well.The following day, the growth medium was removed from the 6-well plates and cells were washed with DMEM followed by addition of the growth medium.Then, the cells were transfected with 4 µg of S protein DNA using Lipofectamine 2000 transfection kit.Vero E6-TMPRSS2-GFP 11 cells were plated into 96-well, glass bottom, black-walled plates (CellVis) at a density of 36,000 cells per well.Twenty-four hours after transfection, BHK-21-GFP 1-10 cells expressing the S protein were washed three times using FluoroBrite DMEM (Thermo Fisher) and detached using an enzymefree cell dissociation buffer (Gibco).9,000 BHK-21-GFP 1-10 cells were added to each well with or without TRI2-2 with the 1:4 serial dilution starting from the initial concentration of 70nM, and the mixture was incubated at 37 °C and 5% CO 2 for 2h before being transferred on top of the Vero E6-TMPRSS2-GFP 11 that was washed 3 times with FluoroBrite DMEM.The mixture was incubated at 37 °C and 5% CO 2 in a Cytation 7 plate Imager (BioTek) and both bright-field and GFP images were collected every 30 min for 18 h.Fusogenicity was assessed by measuring the area showing GFP fluorescence for each image using Gen5 Image Prime v3.11 software.Raw grayscale 16-bit images were pseudocolored in ImageJ using Green Hot Look Up Table.

Cryo-EM sample preparation and data collection
The SARS-CoV-2 BA.2.86 S complex with TRI2-2 at a molar ratio of 1:8 just before the grid preparation.The cryo-EM dataset was collected over two different sessions which were combined to be processed together.3 µL of SARS-CoV-2 BA.2.86 S (Acro Biosystems, SPN-C524y) complex with TRI2-2 at 0.6mg/mL was added to a glow discharged (30s at 15mA) UltraAuFoil R1.2/1.3:Au300grid 30 prior to plunge freezing using a vitrobot MarkIV (ThermoFisher Scientific) with a blot force of -1, wait time of 10s, and 6 sec blot time at 100 % humidity and 22°C.3.5 µL of SARS-CoV-2 BA.2.86 S produced following the aforementioned protein production complex with TRI2-2 at 0.2mg/mL was added to a glow discharged (10s at 15mA) Quantifoil 2nm C Au300 grid prior to plunge freezing using a vitrobot MarkIV (ThermoFisher Scientific) with a blot force of -1, 4 sec blot time, and 10s wait time at 100 % humidity and 22°C.Data were acquired using an FEI Titan Krios transmission electron microscope operated at 300 kV and equipped with a Gatan K3 direct detector and Gatan Quantum GIF energy filter, operated in zero-loss mode with a slit width of 20 eV.Automated data collection was carried out using serialEM 31 at a nominal magnification of 105,000x with a pixel size of 0.829 Å.The dose rate was adjusted to 53e -/Å 2 , and each movie was acquired in counting mode fractionated in 79 frames of 50ms for UltraAuFoil and 99 frames of 40ms for Quantifoil dataset, respectively.A total of 20,217 and 15,703 micrographs were collected for each datasets, respectively.Stage was tilted 0, 30, and 45 degrees for collection with the UltraAuFoil grid.

Cryo-EM data processing, model building and refinement
Particles were extracted with a box size of 320 pixels with a pixel size of 1.658 Å using WARP.Two rounds of reference-free 2D classification were performed using CryoSPARC 32 to select well-defined particle images from each dataset.Particles belonging to classes with the best resolved spike protein density were selected.To improve particle picking further, we trained the Topaz 33 picker on Warp-picked particle sets belonging to the selected classes after 2D classification.The particles picked using Topaz were extracted and subjected to 2D classification using cryoSPARC.The two different particle sets picked from Warp and Topaz were merged and duplicate particle picks were removed using a minimum distance cutoff of 90 Å.Initial model generation was done using ab-initio reconstruction in cryoSPARC and used as references for a heterogenous 3D refinement in cryoSPARC.After two rounds of ab-initio reconstructions and heterogeneous refinements to remove junk particles, 3D refinement was carried out using non-uniform refinement in cryoSPARC 34 and the particles were transferred from cryoSPARC to Relion using pyem 35 (https://github.com/asarnow/pyem) to be subjected to the Bayesian polishing procedure implemented in Relion 36 during which particles were reextracted with a box size of 512 pixels and a pixel size of 1.0 Å.After ab-initio reconstructions and heterogeneous refinements to select best class, subsequent 3D refinement used nonuniform refinement along with per-particle defocus refinement in cryoSPARC to yield the final reconstruction at 2.4 A resolution comprising 811,069 particles.To further improve the density at the RBD:TRI2-2 interface, 3D classification and local refinement was performed using Relion and cryoSPARC with a soft mask comprising the RBD and TRI2-2 yielding a reconstruction at 3.2 Å resolution enabling model building.Reported resolutions are based on the 0.143 goldstandard Fourier shell correlation (FSC) criterion and Fourier shell correlation curves were corrected for the effects of soft masking by high-resolution noise substitution 37,38 .To further improve the N terminus domain (NTD) density, particles belonging to the best selected classes were subjected to another round of heterogeneous refinement, followed by non-uniform refinement with per-particle defocus.Particles were then symmetry expanded following C3 axis and local refinement was performed using cryoSPARC with a soft mask comprising the NTD domain yielding a reconstruction at 2.8 Å resolution enabling model building.UCSF Chimera, UCSF ChimeraX, and Coot were used to fit and rebuild atomic models into the cryoEM maps utilizing sharpened and unsharpened maps.The models were refined and relaxed using Rosetta 39,40 and validated using Phenix 41 , Molprobity 42 and Privateer 43 .Vero-TMPRSS2-hACE2 cells were seeded at a density of 1 × 10 5 cells per well in 24-well tissue culture plates.The following day, medium was removed and replaced with 200 µL of material to be titrated diluted serially in DMEM supplemented with 2% FBS.After 1 h, 1 mL of methylcellulose overlay was added.Plates were incubated for 72 h, then fixed with 4% paraformaldehyde (final concentration) in phosphate-buffered saline (PBS) for 20 min.Plates were stained with 0.05% (wt/vol) crystal violet in 20% methanol and washed twice with distilled, deionized water.

Statistical analysis
and Fig S5).These results indicate that intranasal administration of TRI2-2 confers protection against SARS-CoV-2 challenge in a stringent model of disease with three key SARS-CoV-2 Omicron variants.
J.B.C., Y.J.P., R.R., D.B., M.S.D. and D.V. are co-inventors on a patent application that incorporates discoveries described in this article (application no.: PCT/US2021/034069, title: SARS-CoV-2 inhibitors).M.S.D. is a consultant or advisor for Inbios, Vir Biotechnology, IntegerBio, Moderna, Merck, and GlaxoSmithKline.The Diamond laboratory has received unrelated funding support in sponsored research agreements from Vir Biotechnology, Moderna, Emergent BioSolutions, and IntegerBio.Supplementary Figure 1.Kinetic analysis of TRI2-2 binding to SARS-CoV-2 variant RBDs immobilized on using biolayer interferometry.Biotinylated RBDs were immobilized on streptavidin biosensors to a final level of 1 nm shift each.The TRI2-2 concentrations used are provided in the color keys.Dashed black lines represent curve fits obtained using global fitting and a 1:1 binding model in the ForteBio BLI software.Representative graphs are shown from two biological replicates.
Supplementary Figure 2. Dose-response curves for neutralization of SARS-CoV-2 S variant VSV pseudoviruses by the TRI2-2 and AHB2 miniprotein inhibitors.Each dot represents the mean of three technical replicates.SD shown as lines.Representative graphs are shown from three biological replicates.V al challenged with BQ.1.1 (G), or XBB.1.